Protein misfolding is at the basis of many disease processes, due either to the loss of function of the misfolded protein or cell and tissue damage caused by accumulation of aberrant protein structures(eg. amyloid). Cystic fibrosis (CF), in which the most common mutation (AF508 ) in the CFTR chloride channel gene prevents normal conformational maturation and trafficking from the endoplasmic reticulum (ER) to the cell surface, has become a prototype for the loss of function category of folding disease. The AF508 mutation is temperature sensitive and the mutant protein is active when induced to traffic at permissive temperature or by other means. However, the molecular events in the assembly of the wild-type multidomain CFTR protein and the cellular recognition mechanisms that distinguish the mutant from the wild-type remain unknown. Through integration of our skills in CFTR structure/function (Riordan) and protein folding and trafficking (Balch) we have have made considerable progress towards filling these knowledge gaps by developing new experimental tools for study of the system, mapping of key domain interactions in CFTR assembly, discovery of an acidic ER export code that must be exposed for recognition by the COP II coat on budding ER vesicles and identification, by proteomic methods, of chaperone complexes on both sides of the ER membrane that contribute to the formation of a mature structure . Significantly, we have found that manipulations of the levels of cytoplasmic Hsp90 cochaperones strongly influence the nascent AF508 polypeptide. Reduction of the level of one member of this complex, Aha 1 results in substantial rescue of the mutant protein. Therefore our aims in the continuing work are 1) to elucidate the steps in normal CFTR domain assembly and their perturbation by AF508 and 2) to define the role of the chaperone environment in ER export of CFTR to enable AF508 export by manipulation of this environment. Our joint efforts will test the central hypothesis that the primary impact of the AF508 mutation is to prevent an explicit set of sequential domain assembly steps which rely on spatially and temporally ordered interactions with molecular chaperone and cochaperone complexes. Public Health Relevence- Learning how the assembly and movement of the CFTR ion channel to the cell membrane occurs and manipulation of the steps defective in CF patients will enable development of new therapeutic interventions and provide insight into other major folding diseases.